ORIGINAL�ARTICLE ABSTRACT Objective: To check the expression pattern of cyclin-D1 in stage I head and neck squamous cell carcinoma (H.N.S.C.C.) cases (evaluated based on history, clinical, medical, and radiological records) in our setting and evaluate the role of cyclin-D1 as a prognostic marker of H.N.S.C.C. (considering recurrence within five years and metastasis). Study Design: Retrospective cohort study. Place and Duration of Study: The study was carried out at Armed Forces Institute of Pathology (A.F.I.P.) Rawalpindi working in conjunction with the Department of Radiation Oncology, Rawalpindi. The duration of th th study was from 7 March 2022-14 October 2022. Materials and Methods: The study comprised 92 patients from year 2015-16 of stage I diagnosed with squamous cell carcinoma of head and neck with complete five-year follow-up after fulfilling the inclusion and exclusion criteria. After collecting and matching the data on follow-up with record of the patients from Department of Radiation Oncology, C.M.H., Rawalpindi; paraffin-embedded blocks of these patients from year 2015/2016 were retrieved. Fresh Hematoxylin and Eosin (H&E) slides were prepared, and diagnosis was reconfirmed, vimentin stain was applied to check the antigenicity of the tissue followed by application of cyclin- D1 immuno-histochemical marker (I.H.C.) marker with controls to check the expression pattern. Results: In our study we included a total of 92 diagnosed cases of stage I H.N.S.C.C. with a complete 5-year follow-up. Out of 92 cases male to female ratio was almost 3:1 with males comprising a total of 68 (73.9%) and females comprising 24 (26.1%). Mean age of patients was 62.48+ 11.439 years. Larynx was the most frequently involved site 34.8% (n=32), followed by tongue 23.9% (n=22) and buccal mucosa 13.0% (n=12). Cyclin-D1 expression and tumor grade were revealed to have a statistically significant clinicopathological relationship (p value 0.001). A statistically significant association between the expression of cyclin-D1 and tumor recurrence (p value < 0.001) and duration of tumor recurrence (p value 0.003) was seen. The cyclin-D1 expression was also compared with metastasis which was also statistically significant (p value 0.003). Conclusion: An increased risk of metastasis and recurrence is linked to increased levels of cyclin-D1 expression in the early years. Hence it is valuable to include cyclin-D1 expression in the initial diagnostic work up of H.N.S.C.C. for an early prognostic assessment. Further use of these results could be made for targeted treatment of head and neck malignancies. Key Words: Cyclin-D1, Five-Year Follow-Up, Head-and-Neck Squamous Cell Carcinoma, Immuno-Histochemical Expression, Prognosis. with distinct clinical and biological characteristics, causing an estimated 17,960 deaths worldwide each 1 year. H.N.S.C.C. has a substantial risk of both metastases and local recurrence, so aggressive treatments are typically performed to maximize the 2 likelihood of long-term control. These treatments may include surgery, radiotherapy, and/or chemotherapy. The high prevalence of this cancer requires special concern to devise authentic treatment protocols that are least invasive and show 3 better outcomes. Despite advancements in cancer prevention and treatment, the five-year survival rate of a patient with head and neck squamous cell Introduction Among the top ten types of human cancer, head and neck malignancies are a diverse category of tumors Prognostic Value of Cyclin-D1 Expression in Head-a nd-Neck Squamous Cell Carcinoma: A Half-Decade Follow-Up 1 2 3 4 5 6 Azka Haroon , Muhammad Nadeem Zafar , Nighat Ara ,Saadia Muneer ,Zunaira Saeed , Zainab Asif Sukhera Correspondence: Dr. Azka Haroon Department of Histopathology Armed Forces Institute of Pathology, Rawalpindi E-mail: azkaharoon.haroon@gmail.com 1,2,4,5,6 Department of Histopathology Armed Forces Institute of Pathology, Rawalpindi 3 Department of Oral pathology, Army Medical College, Rawalpindi Received: December 18, 2022; Revised: March 13, 2023 Accepted: March 14, 2023 Prognostic Value of Cyclin-D1 in HNSCCJIIMC 2023 Vol. 18, No.1 22 https://doi.org/10.57234/jiimc.march23.1600 carcinoma remains lower than that of other malignancies, such as colorectal, breast and 4 cervical. In conventional T.N.M. classification systems, the molecular heterogeneity of squamous cell carcinoma of the head and neck (H.N.S.C.C.) is 5 not taken into account. Hence, it is imperative to understand how recent molecular markers that have been identified correlate with prognosis for H.N.S.C.C., as they may offer new methods for early diagnosis, evaluation of prognosis and treatment. Cyclin-D1 a gene found on chromosome 11q13 plays 6 a major role in activation of cell-cycle progression. Cyclin-D1 overexpression has been proposed by various international studies as a good marker for predicting poor prognosis in diagnosed cases of 6,7,8,9 H.N.S.C.C., but these studies show the lack of proper follow-up, the uncertainty of such results in oral squamous cell carcinoma (O.S.C.C.), and smaller 10 overall sample size. This study's objective was to assess the expression of cyclin-D1 on a larger sample of stage I H.N.S.C.C. cases with a documented complete five-year follow-up to determine its prognostic significance. So that if the association is proved then cyclin-D1 marker should be made part of the diagnostic protocol. Patients with strong expression of cyclin-D1 on premier biopsy should be kept at close follow-up and treatment should be planned accordingly (targeted therapy can be given to patients such as cyclin-D inhibitors). If on the contrary this association is disapproved then we should stop investing time and money on this marker and evaluate better prognostic markers for the improvement in the treatment of patients with H.N.S.C.C. Information in regard to this study was collected on data collection questionnaire in the form of variables, statistical analysis was performed using S.P.S.S. version 26.0, Microsoft Excel 2013 was used for diagrammatic representations, qualitative / categorical variables were presented as frequency and percentages, to compare various parameters Chi-square test was performed and p-value of 0.05 was considered to be the essential level of significance. Materials and Methods A retrospective cohort study was carried out at Armed Forces Institute of Pathology (A.F.I.P.). This study had 92 patients in total. The approach of non- probability convenient sampling was applied. The study was conducted after the approval of Ethical Review Committee (Letter number: MP-OMP20- th 1/READ-IRB/21/781, dated: 30 -December-2021). Contact details of all cases diagnosed from Armed Forces Institute of Pathology (A.F.I.P.) of squamous cell carcinoma of head and neck region from January 1, 2015, to December 31, 2016 were taken from archives of Histopathology Department at Armed Forces Institute of Pathology (A.F.I.P.), Rawalpindi, Pakistan. The diagnosed patients of stage 1 H.N.S.C.C. were contacted for consent to participate in the research and five-year follow-up i.e., till December 31, 2021, and onwards. The data was matched with the record of these patients kept in the Department of Radiation Oncology, C.M.H. Rawalpindi. After collecting the data on follow-up of these patients, paraffin-embedded blocks of these patients from year 2015 to 2016 were retrieved. Confounding factors were excluded by firmly following the exclusion criteria [patients lost to follow-up or could not be contacted, poorly fixed specimens, very scanty tissue specimens, extensive necrosis or the retrieved blocks failed to take vimentin stain to check the antigenicity of tissue, patients dying within the study period, patients who had irretrievably taken away paraffin sections, cases diagnosed with H.N.S.C.C. of conjunctiva or skin or cases diagnosed with any immuno-morphological subtype of squamous cell carcinoma or mixed forms (with more than one component) i.e. adeno- squamous carcinoma ] and inclusion criterion [all stage I H.N.S.C.C. cases with a documented complete five-year follow-up (evaluated on the basis of history, clinical, medical and radiological records) in our setting at A.F.I.P. during the calendar year January 1, 2015- December 31, 2016 (irrespective of age, gender or ethnicity)].H&E slides were prepared freshly for already diagnosed cases, but to eliminate bias the slides were viewed by three investigators separately. After confirmation of diagnosis on freshly prepared H&E slides, vimentin stain was applied to check the antigenicity of the tissue, followed by the application of cyclin-D1 immunohistochemical marker by Leica Microsystem (Germany). Lymph node tissue (mantle cell lymphoma) was used as positive control and appendix tissue was used as negative controls for cyclin-D1. When distinct brown nuclear staining was observed in ≥1% of the cells, the Prognostic Value of Cyclin-D1 in HNSCCJIIMC 2023 Vol. 18, No.1 23https://doi.org/10.57234/jiimc.march23.1600 tissue samples was considered positive for cyclin-D1. Cytoplasmic staining was not considered. The quantification criteria by Dhingra et al., was used to 9 quantify cyclin-D1 expression. The sections with an inter-observer difference of more than 10% were re- examined by means of a multi-headed light microscope to reach consensus. Information was collected on data collection questionnaire in the form of variables, statistical analysis was performed using S.P.S.S. version 26.0, Microsoft Excel 2013 was used for diagrammatic representations, qualitative / categorical variables were presented as frequency and percentages, to compare various parameters chi-square test was performed and p-value of 0.05 was the essential level of significance. Results A total of 92 diagnosed cases of stage I H.N.S.C.C. with a complete 5-year follow-up were included in the study. Out of 92 cases male to female ratio was almost 3:1 with males comprising a total of 68 (73.9%) and females comprising 24 (26.1%). Mean age of patients was 62.48+ 11.439 years. Larynx was the most frequently involved site 34.8% (n=32), followed by tongue 23.9% (n=22) and buccal mucosa 13.0% (n=12). The chi-square test revealed a stat i st i ca l l y s i g n i f i ca nt c l i n i co - p at h o l o g i ca l relationship between cyclin-D1 expression and tumor grade (p value 0.001) Table I. The cyclin-D1 expression was also compared with recurrence within five years by using chi-square test which was statistically significant (p value < 0.001) Table II and a statistically significant association was also seen between cyclin-D1 expression and duration of tumor recurrence (p value 0.003) Table III. The cyclin-D1 expression was also compared with metastasis within five years by using chi-square test which was also statistically significant (p value = 0.003) Table IV Discussion Cyclin-D1 is a gene located on chromosome 11q13 and plays a major role in activation of cell-cycle progression. Cyclin-D is a cyclin-dependent kinase (C.D.K.) 4 and C.D.K. 6-binding cell cycle regulator. The stimulation of D.N.A. synthesis and the transition of cells between the G1/S phases are both 11 significantly influenced by this cyclin-CDK complex, 12 rendering it a genuine target during carcinogenesis. As a result, it has been determined that 30% of head 13 and neck S.C.C. have cyclin-D1 amplification. In our investigation, it was discovered that cyclin-D1 expression positively associated with tumor grade. When the histopathological grade increased, from well differentiated to poorly differentiated Table I: Case Distribution Based on Overall Cyclin-D1 Expression Score and Tumor Grade (n=92) p-value = 0.001 Table II: Case Distribution Based on Overall Cyclin-D1 Expression Score with Recurrence (n=92) p-value = 0.001 Table III: Case Distribution Based on Overall Cyclin-D1 Expression Score With Recurrence as Per Follow up Duration (N=92) p-value = 0.003 Table IV: Case Distribution Based on Overall Cyclin-D1 Expression Score with Metastasis (n=92) p-value = 0.003 Prognostic Value of Cyclin-D1 in HNSCCJIIMC 2023 Vol. 18, No.1 24 https://doi.org/10.57234/jiimc.march23.1600 H.N.S.C.C., the percentage of cyclin-D1 expression increased as well. In various studies, higher cyclin-D1 expression has been linked to poor histological 8 grade. According to Chinnathambi et al., in year 2021cyclin-D1 immuno-expression was discovered in all patients, and it was substantially correlated with deteriorating tumor grade and positive lymph node disease. Nevertheless, several authors discovered no clinical correlation. Batool et al. in 2020 in her study described similar findings that the histological grade of H.N.S.C.C. was not substantially 14 correlated with cyclin-D reactivity. Zand et al. in 2020 and Nazar et al. in year 2020 also described that the results of their study demonstrated no link between expression of cyclin-D1 and grade of the 15, 16 disease. The main finding of our research was that the stage I diagnosed cases of H.N.S.C.C. showing an elevated expression of cyclin-D1 at the time of diagnosis were at a greater risk of developing recurrence in the earlier years of five-year period of follow-up as compared to those cases showing a weak or no expression of cyclin-D1 immuno-histochemical stain. Another important finding of this study was that cyclin-D1 expression was found to positively correlate with tumor metastasis. An increase in cyclin-D1 expression at the time of diagnosis of stage I cases of H.N.S.C.C. was found to show an increased risk of developing tumor metastasis within the five year of diagnosis of tumor. Various studies have been conducted on this molecular marker suggesting that strong positivity of cyclin-D1 expression in H.N.S.C.C. is related to metastasis and recurrence in operable cases of 9 H.N.S.C.C. Sharada et al., in year 2018 and Patel et al., in year 2017 described similar results in their studies that overexpression of cyclin-D1 has been seen to associate with an increased risk of metastasis 17,18 of lymph nodes and increased risk of recurrence. Our research had this limitation that five year survival of the cases could not be included in the study to determine prognostic value of cyclin-D1 as it was a retrospective study and confounders could not be removed for those who died within five years; as exact cause of death could not be determined for those dying within study period (five years of diagnosis of H.N.S.C.C.).Hence, prospective follow- up study is recommended including five year survival of cases as a variable to determine prognostic value of cyclin-D1 thus minimizing confounders by notifying exact cause of death of participants within the study period. Conclusion This study showed a statistically strong association between recurrence, duration of developing recurrence and metastasis with increasing cyclin-D1 expression. Therefore, an increased risk of metastasis and recurrence is linked to increased levels of cyclin-D1 expression in the early years. Hence it is valuable to include cyclin-D1 expression in the initial diagnostic work up of H.N.S.C.C. for an early prognostic assessment. . Further use of these results could be made for targeted treatment of head and neck malignancies. REFERENCES 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of This figure is showing expression score as per quantification criteria. This figure is showing intensity score as per quantification criteria Figure 1: Figure Showing Total Score of Cyclin-D1 Expression (Product of Expression Score and Intensity Score) Prognostic Value of Cyclin-D1 in HNSCCJIIMC 2023 Vol. 18, No.1 25https://doi.org/10.57234/jiimc.march23.1600 incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68(6): 394-424. DOI: 10.3322/caac.21492. 2. Bhat AA, Yousuf P, Wani NA, Rizwan A, Chauhan SS, Siddiqi MA, et al. Tumor microenvironment: an evil nexus promoting aggressive head and neck squamous cell carcinoma and avenue for targeted therapy. Sig Transduct Target Ther. 2021; 6(1): 12. DOI: https://doi.org/10. 1038/s41392-020-00419-w. 3. Burgener J, Zou J, Zhao Z, Zheng Y, Shen SY, Huang SH, et al. Tumor-naïve multimodal profiling of circulating tumor DNA in head and neck squamous cell carcinoma. Clin Cancer Res. 2021; 7(15): 4230-44. DOI: 10.1158/1078-0432.CCR-21- 0110. 4. Burtness B, Rischin D, Greil R, Soulières D, Tahara M, de Castro Jr G, et al. Pembrolizumab alone or with chemotherapy for recurrent/metastatic head and neck squamous cell carcinoma in KEYNOTE-048: subgroup analysis by programmed death ligand-1 combined positive score. J Clin Oncol. 2022; 40(21): 2321-32. DOI: 10.1200/JCO.21.02198. 5. Girolami I, Pantanowitz L, Barberis M, Paolino G, Brunelli M, Vigliar E, et al. Challenges facing pathologists evaluating PD-L1 in head & neck squamous cell carcinoma. J Oral Pathol Med. 2021; 50(9): 864-73. DOI: 10.1111/jop.13220. 6. Chen Y, Huang Y, Gao X, Li Y, Lin J, Chen L, et al. CCND1 amplification contributes to immunosuppression and is associated with a poor prognosis to immune checkpoint inhibitors in solid tumors. Front Immunol. 2020; 11: 1620. DOI: 10.3389/fimmu.2020.01620. 7. Yu G, Li N, Wang W, Niu M, Feng X. p28GANK overexpression is associated with chemotherapy resistance and poor prognosis in ovarian cancer. Oncol Lett. 2020; 19(1): 505-12. DOI: 10.3892/ol.2019.11081. 8. Moharil RB, Khandekar S, Dive A, Bodhade A. Cyclin D1 in oral premalignant lesions and oral squamous cell carcinoma: An immunohistochemical study. J Oral M a x i l l o f a c i a l P a t h o l . 2 0 2 0 2 4 ( 2 ) : 3 9 7 . D O I : 10.4103/jomfp.JOMFP_164_20. 9. Dhingra V, Verma J, Misra V, Srivastav S, Hasan F. Evaluation of cyclin D1 expression in head and neck squamous cell carcinoma. Journal of clinical and diagnostic research. J Clin Diagn Res. 2017 Feb; 11(2): EC01–EC04. DOI: 10.7860/JCDR/2017/21760.9329. 10. Johnson DE, Burtness B, Leemans CR, Lui VW, Bauman JE, Grandis JR. Author Correction: Head and neck squamous cell carcinoma (Nature Reviews Disease Primers, (2020), 6, 1, (92), 10.1038/s41572-020-00224-3. Nat Rev Dis Primers. 2023 Dec;9(1):4. DOI: 10.1038/s41572-023-00418-5. 11. De Bakker T, Journe F, Descamps G, Saussez S, Dragan T, Ghanem G, et al. Restoring p53 function in head and neck squamous cell carcinoma to improve treatments. Front Oncol. 2022; 11: 1-11. DOI: 10.3389/fonc.2021.799993. 12. Go SI, Ko GH, Lee WS, Lee JH, Jeong SH, Lee YJ, et al. Cyclin D1 serves as a poor prognostic biomarker in stage-I gastric cancer. Curr Issues Mol Biol. 2022; 44(3): 1395-406. DOI: 10.3390/cimb44030093. 13. Kordbacheh F, Farah CS. Molecular pathways and druggable targets in head and neck squamous cell carcinoma. Cancers. 2021; 13(14): 1-21. DOI: 10.3390/cancers13143453. 14. Batool S, Ali SN, Soomro N, Ali SL. Association of cyclin D expression with malignant transformation of oral mucosa in tobacco users. Rawal Med J. 2020; 45(2): 342-46. 15. Huang J, Lok V, Ngai CH, Chu C, Patel HK, Chandraseka VT, Zhang L, et al. Disease burden, risk factors, and recent trends of liver cancer: A global country-level analysis. Liver Cancer. 2021; 10(4): 330-45. DOI: 10.1159/000515304. 16. N a z a r M , N a z I , M a h m o o d M K , H a s h m i S N . Immunohistochemical expression of Cyclin D1 and Ki-67 in primary and metastatic oral squamous cell carcinoma. A s i a n Pa c J C a n c e r P re v. 2 0 2 0 ; 2 1 ( 1 ) : 3 7 - 4 1 . DOI:10.31557/APJCP.2020.21.1.37. 17. Sharada P, Swaminathan U, Nagamalini BR, Vinodkumar K, Ashwini BK, Lavanya V. A semi-quantitative analysis of immunohistochemical expression of p63, Ki-67, Cyclin-D1, and p16 in common oral potentially malignant disorders and oral squamous cell carcinoma. J NTR Univ Health Sci. 2018; 7(2): 120-8. 18. Denaro N, Merlano MC, Lo Nigro C. Further understanding of the immune microenvironment in head and neck squamous cell carcinoma: implications for prognosis. C a n c e r M a n a g R e s . 2 0 2 1 : 3 9 7 3 - 8 0 . D O I : 10.2147/CMAR.S277907. CONFLICT OF INTEREST Authors declared no conflicts of Interest. GRANT SUPPORT AND FINANCIAL DISCLOSURE Authors have declared no specific grant for this research from any funding agency in public, commercial or nonprofit sector. DATA SHARING STATMENT The data that support the findings of this study are available from the corresponding author upon request. This is an Open Access article distributed under the terms of the Creative Commons Attribution- Non- Commercial 2.0 Generic License. Prognostic Value of Cyclin-D1 in HNSCCJIIMC 2023 Vol. 18, No.1 26 https://doi.org/10.57234/jiimc.march23.1600